Detection and mapping of point mutations using partial digestion

ABSTRACT

According to the present invention there is provided a method for detecting the presence of at least two point mutations in a target polynucleotide, as well as their relative positions and specific nucleotide positions, involving partial digestion and the use of mismatch repair enzymes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US98/06878, filed Apr. 15, 1998. Thisapplication also claims priority to U.S. Provisional application No.60/043,184, filed Apr. 16, 1997.

FIELD OF THE INVENTION

The present invention relates to a method for detecting the presence ofat least two point mutations in a target polynucleotide, as well astheir relative positions and specific nucleotide position, via partialdigestion. The present invention further relates to a method fordetecting the presence of at least two point mutations in a targetpolynucleotide, as well as their relative positions and specificnucleotide position via a combination of partial digestion and anoscillation reaction.

BACKGROUND OF THE INVENTION

Genomic DNA provides the template for the information that allows thegeneration of proteins which are expressed and made by an organism.These proteins are generally essential for the survival of any specificcell in an organism. Therefore, the organism requires the template to becorrect and free of mistakes in order to generate a protein that isfunctional in a cell. The protein may be nonfunctional if a singlenucleotide of this DNA sequence is mutated (“a point mutation”). Pointmutations which elicit disease states are known for many proteins.

Recent advances have allowed for the detection of point mutations withmismatch repair enzymes. Hsu et al., Carcinogenesis 15: 1657 (1994),describe the detection of A/G point mutations with mutY repair enzyme.Xu et al., Carcinogenesis 17(2): 321 (1996) further describe using mutYto detect A/G and to a lesser extent C/A mutations.

Youil et al., PNAS 92: 87 (1995) relate techniques for screening foreach of the possible eight point mutations i.e. G/A, C/T, C/C, G/G, A/A,T/T, C/A, and G/T, using T4 endonuclease VII. Lu et al., WO93/20233 at29-30 describe screening for mutations using all-type enzyme, whichrecognizes all eight base pair point mutations. Lu et al. also describethe use of combinations of different repair enzymes to ascertain thepresence of an unknown point mutation in a sample. Id. at 27.

These techniques are directed, however, to the detection of a singlepoint mutation in a given polynucleotide sample. To the extent that anucleic acid target molecule has multiple base pair mutations spanningits length, the above methods are either ineffective or inefficient indetecting the presence and relative position of these mutations.

WO 96/40902 is also a background publication.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a methodfor detecting the presence of at least two point mutations in a targetpolynucleotide, as well as their relative positions and specificnucleotide position via partial digestion.

It is a further object of the present invention to provide a method fordetecting the presence of at least two point mutations in a targetpolynucleotide, as well as their relative positions and specificnucleotide position via a combination of partial digestion and anoscillation reaction.

In accomplishing the foregoing objects as well as other objects, thereis provided a method of detecting the presence of and determining therelative positions of at least two point mutations in targetpolynucleotides, comprising:

(a) hybridizing single-stranded oligonucleotide probes to targetpolynucleotides to form hybrid, double-stranded polynucleotides suchthat mismatches occur at the sites of the point mutations, wherein theprobes are complementary to a non-mutated sequence of the targetpolynucleotides and are labelled-at one end but not both ends, andwherein the target polynucleotides are not labelled;

(b) partially digesting the probe strands of the hybrid polynucleotideswith a nucleic acid repair enzyme such that probe fragments of differinglengths are generated;

(c) separating the probe fragments by size in a medium suitable forvisualizing the separated probe fragments; and then

(d) visualizing the separated probe fragments in the medium, whereby thepresence and relative positions of the point mutations are determined.

There is further provided a method of detecting the presence of anddetermining the relative positions of at least two point mutations in atarget polynucleotide, comprising:

(a) hybridizing a single-stranded oligonucleotide probe to a targetpolynucleotide to form a hybrid, double-stranded polynucleotide suchthat mismatches occur at the sites of the point mutations, wherein theprobe is complementary to a non-mutated sequence of the targetpolynucleotide and is labelled at one end but not both ends, and whereinthe target polynucleotide is not labelled;

(b) partially digesting the probe strand of the hybrid polynucleotidewith a nucleic acid repair enzyme producing oligonucleotide fragments,wherein the oligonucleotide probe is designed such that theoligonucleotide fragments dissociate from the target polynucleotidespontaneously at a predetermined temperature;

(c) repeating steps (a) and (b) such that probe fragments of differinglengths are generated;

(d) separating the probe fragments by size in a medium suitable forvisualizing the separated probe fragments; and then

(e) visualizing the separated probe fragments in the medium, whereby thepresence and relative positions of the point mutations are determined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the detection of three pointmutations using the partial digestion method of the instant invention.Probe is hybridized to target and then nucleic acid repair enzyme isadded, which partially digests the probe. The resulting probe fragmentsare then detected.

FIG. 2 is a schematic diagram showing the detection of two pointmutations using partial digestion in combination with an oscillationreaction. Probe is hybridized to target and then nucleic acid repairenzyme is added, which partially digests the probe. The probe thendissociates from the target, with or without an increase in temperature,and the cycle is repeated.

FIG. 3 is a representation of the DNA sequence of orf10 and the aminoacid sequence of the corresponding ORF10 polypeptide (SEQ ID NOS 9 and10).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The above-mentioned disadvantages of conventional techniques fordetecting multiple point mutations have been overcome by the presentinvention. In particular, it has been discovered that partial digests,effected using mismatch repair enzymes, allow for the detection andmapping of multiple base pair mutations.

Partial digest mapping has been limited heretofore to restrictionendonucleases. For example, see Danna et al., 65 METHODS IN ENZYMOLOGY,Ch. 53, at page 449 (Academic Press 1980). Restriction endonucleases arenot desirable for detecting multiple point mutations, however, because arestriction enzyme can only detect a mutation if it is located withinthe enzyme's specific cleavage recognition site.

The instant invention therefore has overcome the above-mentioneddisadvantages of existing techniques for detecting point mutations. Theinventive approach entails hybridizing single-stranded oligonucleotideprobes to target polynucleotides to form hybrid, double-strandedpolynucleotides. The hybridization preferably occurs under conditionsthat are “stringent,” which typically implicates conditions that includea 50-100 mM salt solution at a temperature of (3N—20° C.), where N isthe number of nucleotides in the oligonucleotide probe.

As for probe design, preferably, the oligonucleotide probe is designednot to have self complementary regions, palindromic regions and theprobe must also have probe specificity. The parameters for probe designcan be found in Lowe et al., Nucl. Acids Res. 18:1757-1761 (1990);Rychlik et al., loc. cit. 17:8543-8551 (1989); Rychlik et al., loc. cit.18:6409-6412 (1990), which discusses probe design as applied to PCRreactions.

The probe may be a synthetic oligonucleotide, or may be derived from thePCR amplification of a desired genomic DNA or cDNA sequence byamplifying the desired sequence with two primers one of which islabelled. The probe may also be a restriction fragment generated fromrestriction endonuclease cleaving of a desired genomic DNA or cDNAsequence.

The oligonucleotide probe also is labelled. Probe labelling allows forthe detection of cleaved oligonucleotide fragments and may beaccomplished by a number of art recognized methods. For example, theoligonucleotide can be tagged with a radioactive label, such as aradiolabelled nucleotide. Alternatively, the probe can be labelled withbiotinylated nucleotides or florescent nucleotides as known to those ofskill in the art.

In the present invention, the oligonucleotide probe is labelled at oneend but not both. Additionally, the target polynucleotide is notlabelled.

Because the probe is complementary to a non-mutated sequence of thetarget polynucleotide, there will be mismatches between non-mutatedprobe and mutated target polynucleotides at each site of point mutation.In the instant invention, the target polynucleotide will have at leasttwo mismatch sites.

The probe strands of the hybrid probe-target polynucleotides are thenpartially digested with a “nucleic acid repair enzyme,” such that probefragments of differing lengths are generated. The probe fragments arethen separated by size in a medium suitable for visualizing theseparated fragments. The separated fragments are then visualized, andthe presence and relative position of the point mutations aredetermined.

In a further embodiment, the size of the separated probe fragments aremeasured and thereby the specific nucleotide position of each pointmutation is determined. Methods for measuring the size of nucleotidebands in visualizing medium are known to those of skill in the art. Forexample, a size marker can be used to determine the size of a nucleotideband in an electrophoresis gel.

The phrase “polynucleotide target” is used here to denote a nucleotidesequence of any size, including a sequence of less than 30 base pairs,or up to several hundred base pairs. For instance, examples ofpolynucleotide targets include the genes or portions of genes which havebeen isolated from genomic or CDNA libraries by methods known to theskilled artisan, such as PCR amplification. In particular, genes ofinterest would be those known to exhibit point mutations elicitingdisease states. Examples include sickle cell anemia hypoxanthinephosphotransferase and p53, a tumor suppressor gene, as well as severaloncogenes and cancer genes. Other illustrations of polynucleotidetargets include cDNAs generated by reverse transcriptase, and a specificdeoxyoligonucleotide primer encompassing a short sequence immediatelydownstream of a hotspot for mutations within a gene such as BRCA1.

Other examples of polynucleotide targets include cDNAs prepared byreverse transcriptase using mRNA as template and a deoxynucleotideprimer specific for the mRNA of interest. In this case the cDNA wouldact as a target for a labelled oligonucleotide to generate a basemismatch or several base mismatches upon hybridization. The addition ofDNA mismatch repair enzymes would result in the formation of cleavedproducts whose intensity could allow for the quantitation of the mRNA orto determine if the genomic DNA encoding this mRNA has one or more pointmutations.

In fact, this procedure can act as an endogenous method for amplifying atarget gene, such as BRCA1, in that a cell's own production of mRNAproduces a desired amount of target polynucleotide for a partialdigestion assay. Detection of point mutations within target cDNAs bythis embodiment can therefore obviate the need for amplification by PCR.

Another example of a polynucleotide target includes bacterial 16S rRNAgenes and intergenic regions. See Brow et al., J. Clin. Microbiol.34:3129-3137 (1996). In this scenario, a 350 bp segment of E. coli 16SrRNA DNA is amplified by PCR using a labelled primer and an unlabelledprimer. The labelled PCR product, labelled at one end on one of thestrands, is mixed with a target DNA from an unknown bacterium comprisinga PCR product generated with the same unlabelled primers. Denaturationand renaturation of the target and probe DNAs generates a proportion ofmolecules in which the labelled strand comes from the E. coli and theother strand comes from the unknown bacterium. A number of basemismatches are created between the two strands which are cleaved by theDNA mismatch repair enzymes. The cleaved products are detected on adenaturing polyacrylamide gel or by other methods known to the skilledartisan. The pattern of the cleavage products would be characteristic ofthe sequence of the unknown bacterium's 16S rRNA gene and couldtherefore be a means of identification of the unknown organism.

In the present description, the phrase “nucleic acid repair enzyme”denotes an enzyme that cleaves, at a point of mismatch, one strand of aduplex formed by oligonucleotide probe and target polynucleotide.Examples of nucleic acid repair enzymes which can be used in the aboveprocess are mutY (Wu et al., Proc. Nat'l Acad. Sci. USA 89: 8779-83(1992)), T/G mismatch-specific nicking enzyme from HeLa nuclear extracts(Wiebauer & Jiricny, Nature 339: 234-36 (1989); Wiebauer & Jiricny, loc.cit. 87: 5842-45 (1990)), T/G mismatch-specific nicking enzyme from E.coli (Hennecke et al., Nature 353:

776-78 (1991)), human yeast all-type enzymes (Yeh et al., J. Biol. Chem.2667: 6480-84 (1991); Chiang & Lu, Nuc. Acids Res., 19:4761-4766(1981)), Deoxyinosine 3′-Endonuclease from E. coli (Yao et al., J. Biol.Chem. 270: 28609-16 (1995); Yao et al., J. Biol. Chem. 269: 31390-96(1994)).

Another example of nucleic acid repair enzyme is an enzyme systemcomprising a glycosylase combined with an AP cleaving enzyme, such asendonuclease or lyase. Together glycosylase and AP cleaving enzyme, suchas endonuclease or lyase cleave oligonucleotide probe/targetpolynucleotide duplex at a point of mismatch. A glycosylase creates anabasic sugar (an AP site) at the point of mismatch, which then iscleaved by an AP cleaving enzyme, such as endonuclease or lyase.Illustrative enzymes in these categories are detailed below:

I. glycosylases—tag-1, alkA, ung, fpy, muty, nth, xthA, nfo, recJ, uvtA,uvrD, mfd, mutH, mutL, mutS, uracil DNA glycosylase, hydroxymethyluracilglycosylase, 5-mC DNA glycosylase, hypoxanthine DNA glycosylase, thyminemismatch DNA glycosylase, 3-mA DNA glycosylase, hydrated thymine DNAglycosylase (endonuclease III), pyrimidine dimer glycosylase. Theseenzymes can come from any different biological sources. For example,Friedberg et al., DNA REPAIR AND MUTAGENESIS (ASM Press 1995), listsuracil DNA glycosylases from herpes simplex virus types 1 and 2, equineherpes virus, Varicella zoster virus, Epstein Barr virus, humancytomegalovirus, Mycoplasma lactucae, E. coli, B. subtilis, M. luteus,B. steorophermaophilus, Thermothrix thirpara, S. pneumoniae,Dictyostelium discoideium, Artenia salina, S. cerevisae, Hordeumvulgare, Zea mays, Triticum vulgare, rat liver mitochondria, calfthymus, human placenta, HeLa S3 cells, and acute leukemia blast cells.

II. AP cleaving enzymes—E. coli exonuclease III, E. coli endonucleaseIV, Saccharomyes AP endonuclease, Drosphila melanogaster AP endonucleaseI and II, human AP endonuclease, human AP lyase, BAP endonuclease, APEXendonuclease, HAP1 and AP endonuclease.

In addition to the above systems, cleavage may also be effected by usinga glycosylase enzyme, as described above, in combination with basicconditions and increased temperature. In this embodiment, increasing pHand temperature effectuates cleavage at the AP site created by theglycosylase enzyme. Suitable parameters for cleavage of the AP site arepH levels of approximately 8 to 14, and temperatures ranging fromapproximately 50° to 95° C.

In another embodiment, the present invention employs a nucleic acidrepair enzyme that is thermally stable, in the sense that the enzymewould function at some elevated temperature, such as from 50° to 80° C.Additionally, it is preferable that the thermally stable nucleic acidrepair enzyme withstand temperatures up to 100° C. for short periods.

For instance, the present invention contemplates the use of a thermallystable glycosylase. An example of a thermally stable glycosylase is theORF10 protein encoded by the DNA sequence of FIG. 3. This enzyme hasbeen synthesized by Richard P. Cunningham at the State University of NewYork at Albany, according to the methods of Example 4. See also Horst etal., EMBO J. 15: 5459 (1996).

The substrate activity of the ORF10 enzyme includes both base cleavingproperties and AP endonuclease activities. The AP endonucleaseactivities of this enzyme may be enhanced, however, by changing theamino acid residue in position 126 of FIG. 1 from a tyrosine to alysine. This substitution may be achieved by site directed mutagenesisby the methods discussed in Deng, et al., J.A. Anal. Biochem. 200:81(1992).

The ORF10 glycosylase is a homologue of the endonuclease III family. Assuch, the skilled artisan may identify and isolate genes of theendonuclease III family from other thermophilic bacteria. Suitableprobes may be designed as degenerate nucleotide coding sequences for thefollowing amino acid sequences which are highly conserved amongst themembers of the endonuclease III family: (SEQ ID NO:1), PYVILITEILLRRTT;(SEQ ID NO:2), AILDLPGVGKYT; (SEQ ID NO:3), MVDANFVRVINR.

These degenerate oligonucleotides may be used as PCR primers to amplifyportions of the gene from the chromosomal DNA of thermophilic bacteriaby PCR. Such amplified PCR products may then be used to screen a libraryof the thermophilic bacterium. Positive clones would be sequenced andthe coding sequence for the mismatch glycosylase cloned into anexpression vector for protein production.

Additionally, the present invention can utilize a combination of nucleicacid repair enzymes. For example, a nucleic acid repair enzyme can beused in combination with a AP cleaving enzyme. Advantageously, mutY isused in combination with AP cleaving enzymes, such as DNA lyase or DNAAP endonuclease. Such a system of enzymes enhances the speed at whichcleavage occurs.

As for the method step of partially digesting probe strands, partialdigestion techniques are known to those skilled in the art. See Danna,supra. For the purposes of the instant description, a partial digestiondenotes a situation where a nucleic acid does not effectuate completecleavage of all probes stands in a reaction.

For example, where there are two points of mismatch on the probe-targethybrid, a partial digestion with a nucleic acid repair enzyme will notcleave every probe-target hybrid at both points of mismatch. Instead,certain hybrids will be cleaved at the first point of mismatch, otherswill be cleaved at the second point of mismatch, and others still may becleaved at both points.

A partial digestion can be established by various techniques known tothose of skill in the art. For example, a partial digestion is obtainedby limiting the reaction time so that the nucleic acid repair enzymedoes not cleave at all possible recognition sites within the probeoligonucleotide. Other methods for establishing a partial digestioninclude serial dilutions of a reaction solution containing the enzyme orvariation of the cation concentration in the solution.

The cleaved fragments resulting from the above-described partial digestsare separated by size and visualized by methods known to those of skillin the art. Such methods include gel electrophoresis and capillaryelectrophoresis as described above. The length of the cleaved fragmentscan be measured and, and thereby the specific nucleotide position ofeach point mutation determined, by comparing the probe fragments tolabelled DNA fragments of known size, as mentioned above.

In one embodiment of the present invention, the target polynucleotidesand oligonucleotide probes have been amplified by techniques known tothe skilled artisan, such as PCR, so that there is a sufficient amountof probe fragments to be visualized, after the probe fragments have beenseparated by size in a visualizing medium.

In another embodiment of the present invention an oscillation reactionis employed to obviate or diminish the need for amplified targetpolynucleotide. In particular, an oscillation reaction is createdwhereby a nucleic acid repair enzyme partially digests theoligonucleotide probe producing probe fragments which dissociate fromthe target polynucleotide at a predetermined temperature. That is, theoligonucleotide probe is designed so that, at the predeterminedtemperature, the oligonucleotide fragments dissociate from the targetpolynucleotide after cleavage by nucleic acid repair enzyme. A cycle oroscillation reaction then occurs because the target polynucleotidehybridizes to another oligonucleotide probe, and the cleavage process isrepeated.

As a consequence, a small number of target polynucleotides can bedetected in a sample, since a single target polynucleotide catalyses theformation of a large number of oligonucleotide probe cleavage fragments.The oscillation reaction can detect from 10-100 target polynucleotidemolecules in a sample. Theoretically, the oscillation reaction maydetect as little as one target polynucleotide molecule in a sample.

To accommodate the oscillation reaction, a high concentration ofoligonucleotide probe is utilized. In this regard, a suitableradiolabelled probe concentration is from 0.01 to 10 pmol. Otherconcentrations can be used depending on the desired length ofautoradiograph exposure times.

One of skill in the art can refer to Duck et al., BioTechniques 9(2):142 (1990), which refers to CPT a similar but less advantageoustechnique for amplifying probe.

In one embodiment, the oscillating reaction is performed isothermally,i.e., the predetermined temperature of dissociation is approximately thesame as, i.e., within a few degrees of, the temperature ofhybridization. In a preferred embodiment, this isothermal temperature is3N—20° C., here N is the length of the probe in base pairs. Within thisworking range the optimal temperature is determined empirically.Preferably, the reaction is performed with 0.01 to 10 pmol of labeledprobe, in the presence of either synthetic target sequence or DNApurified from a sample source. This target DNA will ranges from 1 to10¹² molecules.

In another embodiment, the oscillation reaction is not carried outisothermally, but instead results from temperature cycling. In thisembodiment, the hybridization is effectuated at a temperature which islower than the temperature of dissociation. In other words, after probehas hybridized to the target polynucleotide, the reaction temperature israised to a predetermined temperature to effectuate the dissociation ofthe probe fragments from the target polynucleotide. The reaction is thencooled to allow non-cleaved probe to hybridize to the targetpolynucleotide, and the cycle is repeated. At this stage, more nucleicacid repair enzymes can be added if needed, to the extent that theoriginal enzymes have lost activity due to the increase of temperature.In a preferred embodiment, the temperature is raised to between 85° C.and 95° C. for 1-2 minutes to dissociate the cleaved probe fragmentsfrom the target polynucleotide. The reaction is then slowly cooled toapproximately 20° C. to allow more non-cleaved probe to hybridize to thetarget polynucleotide, and the cycle is repeated.

In this preferred embodiment, an example of a preferred nucleic acidrepair enzyme is thermophylic thymine DNA glycosylase, in particular,the enzyme is one encoded by the orf10 sequence of FIG. 3. This enzymewill survive several cycles of exposure to 85° C. for short periods.

In either of the above described isothermal or non-isothermalembodiments, hybridization can be facilitated by a helix destabilizingmolecule. For instance, a helix destabilizing molecule can allowhybridization of a 20-mer synthetic oligonucleotide to targetpolynucleotide at 40° C.

By reducing the temperature necessary to achieve hybridization ofoligonucleotide probe to target polynucleotide, helix destabilizingmolecule can reduce the need for thermostable enzymes and expensivethermocyclers.

Exemplary helix-destabilizing molecules include *I, herpes simplexvirus-type I ICP8, nucleolin, and adenovirus DNA-binding protein. SeeTopal & Sinha, J. Biol. Chem. 258(20): 12274-79 (1983); Alberts & Frey,Nature 227: 1313-18 (1970); Hosoda & Moise, J. Biol. Chem. 253(20):7547-55 (1978); Ghisolfi et al., loc. cit., 267(5): 2955-59 (1992);Boehmer & Lehman, J. Virol. 67(2): 711-15 (1993); Zijderveld & van derVleit, J. Virol. 68(2): 1158-64 (1994); Monaghan et al., Nucleic AcidsResearch 22(5): 742-48 (1994).

When facilitated by helix-destabilizing molecule, hybridization inaccordance with the present invention can be effected with long-synthetic oligonucleotides, without the use of thermostable enzymes orexpensive thermocyclers. A “long” oligonucleotide in this context isgreater than 25 nucleotides but preferably not greater than 100nucleotides. Use of such long oligonucleotides affords the advantage ofhybridizing to the target polynucleotide with increased specificity.

The presence of a helix-destabilizing molecule thus allows for the useof long synthetic oligonucleotides, without thermostable enzymes orexpensive thermocyclers. The helix-destabilizing molecule allows for thedispensation of thermostable enzymes because it lowers the temperaturenecessary for hybridization.

The following examples merely illustrate the invention and, as such, arenot to be considered as limiting the invention set forth in the claims.

EXAMPLE 1 Partial Digestion Detection and Mapping of Multiple PointMultiple Point Mutations within a DNA Sequence

The following synthetic oligonucleotides WT CS and MUT NCS (SEQ ID NO:4and SEQ ID NO:6) were synthesized using standard phosphoramiditechemistry, well known to those in the art.

5′-AAATGGAGTTATTCCAACAGATAAAGTGTTGAATGGAATACTTAGTTATCTTGGAATGACTAAAGTAGAATTAGA-3′(WT CS)3′-TTTACCTTAATAAGGTTGTATATTTCACAATTTACCTTATGAATTAATAGAACCTTAATGATTTCATTTTAATCT-³²P(MUT NCS)          I          II         III           IV            V        VI

5′-³²P-labeled wild type noncoding strand (WT NCS) or 5′-³²P-mutatednoncoding strand (MUT NCS) was annealed to the unlabeled wild typecoding strand (WT CS). Mismatched bases in the NCS are underlined andnumbered. Various amounts of thymine DNA glycosylase enzyme (Tdg), whichremoves the T at T/G mismatches or mutY enzyme, which cleaves thephosphodiester bond after the A at A/G mismatches were added andincubated for 1 hr at 55° C. and 37° C. for the Tdg and mutY enzymes,respectively. In some cases, E. coli Endo IV was added to a set of Tdgenzyme tubes to facilitate phosphodiester bond cleavage at T/Gmismatches where the Tdg enzyme has removed the T. At the end of theincubation, the DNA was electrophoresed on a 20% denaturingpolyacrylamide gel. The gel was exposed to X ray film for variousperiods of time.

With a short exposure, the Tdg enzyme detected T/G mismatches I and III.Mismatches III, IV, and VI were detected following a longer exposure.

EXAMPLE 2 Partial Digestion Detection and Mapping of Multiple PointMultiple Point Mutations within a DNA Sequence

The following 75 base sequence of a mutated noncoding strand (NCS) (SEQID NO:5) was synthesized which generates two A/G and two T/G mismatcheswhen hybridized to a wild type coding strand (CS):

5′-TCTAATTCTACTTTAGTAATTCCAAGATAATTAAGTATCCATTAAACACTTTATTTGTTGGAATAACTCCATTT-3′ MutNCS                     4            3            2          1

The two A/G (numbers 2 and 4) and the two T/G mismatches (numbers 1 and3), generated by hybridization of a labelled 75 base mutant probe (MutNCS) to a wild-type target sequence (WT CS), were recognized by the DNAmismatch repair enzymes muty and Tdg enzyme, respectively, at dilutionsof 1:100 and 1:10, respective temperatures of 55° C. for Tdg and 37° C.for mutY and a 1 hour incubation time. As controls, hybridization of a³²P-labelled wild-type probe WT NCS to the same 75 base wild-type targetWT CS polynucleotide failed to generate any mismatches and, therefore,no cleavage products were found.

EXAMPLE 3 Partial Digestion Detection and Mapping of Multiple PointMultiple Point Mutations within a DNA Sequence Combined with anOscillation Reaction

The following synthetic oligonucleotides, WT CS and MUT NCS (SEQ ID NO:4and SEQ ID NO:6) is synthesized using standard phosphoramiditechemistry, well known to those in the art.

5′-AAATGGAGTTATTCCAACAGATAAAGTGTTGAATGGAATACTTAGTTATCTTGGAATGACTAAAGTAGAATTAGA-3′(WT CS)3′-TTTACCTTAATAAGGTTGTATATTTCACAATTTACCTTATGAATTAATAGAACCTTAATGATTTCATTTTAATCT-³²P(MUT NCS)          I          II         III           IV            V        VI

5′-³²P-labeled wild type noncoding strand (WT NCS) or 5′-³²P-mutatednoncoding strand (MUT NCS) is annealed to the unlabeled wild type codingstrand (WT CS). Mismatched bases in the NCS are underlined and numbered.Thymine DNA glycosylase enzyme (Tdg), which removes the T at T/Gmismatches or mutY enzyme, which cleaves the phosphodiester bond afterthe A at A/G mismatches is added and incubated for 1 hr at 55° C. and37° C. for the Tdg and mutY enzymes, respectively. In some cases, E.coli Endo IV is added to a set of Tdg enzyme tubes to facilitatephosphodiester bond cleavage at T/G mismatches where the Tdg enzyme hasremoved the T. At the end of the incubation, the tubes are incubated at95° C. for 2 minutes to denature the partial cleavage products from theWT CS. The temperature is slowly decreased to 20° C. to allow more³²P-labelled MUT NCS probe to anneal to the target WT CS. More Tdgenzyme or mutY enzyme are added to the appropriate tubes and incubationis continued for 1 hour at 55° C. and 37° C., respectively. The cycle isthen repeated. At the end of the final 1 hour incubation, loading bufferis added to the tubes. The DNA molecules are heat denatured andelectrophoresed on a 20% denaturing polyacrylamide gel. The gel isexposed to X ray film, developed and analyzed.

EXAMPLE 4 Thermostable Enzyme Synthesis Bacteria and Plasmids

Escherichia coli JM109 is available from New England Biolabs of Beverly,Mass. and Escherichia coli BW415 is available from the laboratory of Dr.Richard P. Cunningham at the State University of New York, at Albany,Department of Biological Sciences. A similar strain suitable for thisprotocol is BW434 and it is available from the Coli Genetic Stock Centerat Yale University School of Medicine, New Haven, Conn.

BW415λDE3 was made with a λDE3 lysogenization kit from Novagen Inc. ofMadison, Wis. This integration allowed for the efficient expression ofthe T/G mismatch specific thymine-DNA glycosylase from a T7 RNApolymerase driven promoter in an endonuclease III deficient strain ofEscherichia coli. The expression system was contained on plasmid pET14Bfrom Novagen Inc.

Plasmid pUV2 containing the orf10 coding sequence is available from Dr.Jork Nolling, Wageningen Agricultural University of the Netherlands,Department of Microbiology, Hesselink van Suchtelenweg 4, 6703 CTWageningen, The Netherlands. The pUV2 plasmid contains a portion ofpFV1, including the orf10 coding sequence, cloned into pUC19.

The methods for deriving plasmid pFV1 are disclosed in Nolling et al.,Nuc. Acids Res. 20(24): 6501 (1992). Plasmid pUC19 is available fromSigma Chemical Co., of St. Louis, Mo.

Preparation and Manipulation of DNA

Plasmid DNA was prepared from JM109 by a modified alkaline lysis method.The orf10 coding sequence from pUV2 was cloned into pUC19 via aDraI-EcoRI to HincII-EcoRI ligation using restriction endonucleases andligase (from New England Bio Labs) according to suppliers'recommendations. The gene was PCR mutagenized using Taq Polymerase fromPerkin Elmer Corp. to simultaneously change a TTG start codon to ATG,create a NcoI restriction site (SEQ ID NO:7) (5′ GTG GGG CTG GAT TTC CATGGA TGA TGC TAC TAA T3′ and also a BamHI site (SEQ ID NO:8) (5′ C.GA CGGCCA GTG GAT CCA AGG GGG CTG ATG 3′ outside the gene. These newrestriction sites were used to clone orf10 into pET14B.

Enzyme Induction and Purification

This plasmid was transformed into Escherichia coli strain BW415λDE3 andselected from on ampicillin plates. Twenty four liters of cells weregrown in Tryptone yeast (TY) broth supplemented with ampicilin at 37 Cto an OD₅₉₅=0.5, and induced with 1 mM IPTG for five hours. The cellswere harvested by centrifugation at 17,000×g for 20 minutes to yield100.82 grams of cell paste which was stored at −80 C. The cell pelletwas thawed, and suspended in 504 ml of 50 mM Tris-HCI pH 8.0, 200 mMNaCl, 2.5 mM EDTA 0.1 mM PMSF. The cell suspension was sonicated 5×3minutes with a Branson sonifer, and then stirred on ice for one hour.The sonicate was centrifuged at 48,000×g for 20 minutes and thesupernatant was retained. Five percent polyethelenimine was added to afinal concentration of 0.1% and the suspension was stirred for 1 hourand centrifuged at 48,000×g to give a supernatant with a volume of 500mls. The supernatant was dialyzed against 4 L of 50 mM KPO₄ pH 7.2overnight.

The crude extract was loaded onto an 80 ml SP Sepharose® Fast Flow(Pharmacia Biotech Inc. of Piscataway, N.J.) column, washed with 100 mlof 50 mM KPO₄ pH 7.2 and eluted with a 1 L gradient from 0 to 1M NaCl ata flow rate of 10 ml/min. The protein eluted at 0.6M and acharacteristic yellow color in the fractions indicative of an Fe-Scluster of the protein. The fractions containing the protein were pooledand dialyzed against 50 mM KPO₄ pH 6.6, loaded onto a 5 ml DNA agarose(Pharmacia Biotech Inc.) column, washed with 10 ml of 50 mM KPO₄ andeluted with a 100 ml gradient of 0-1 M NaCl at a rate of 1 ml/min. Theprotein eluted at 0.8M NaCl as determined by the criteria above.Fractions containing the protein were pooled and dialyzed overnightagainst 50 mM KPO₄pH 6.6. The protein was loaded onto a 5 ml SP hightrap column (Pharmacia Biotech Inc.) and eluted with 1M NaCl toconcentrate the protein. The extract was further concentrated to 1.5 mlswith Centriprep® 10 concentrators (Amicon Division, W. R. Grace & Co.,of Danvers, Mass.). At this point the protein was a single band on anoverloaded Coomassie® stained gel and gave an A₄₁₀/A₂₈₀ ratio of 0.295.This protein was active in our T-G mismatch assay. The pure protein wasstored in 50% glycerol at −20 C.

10 1 15 PRT Unknown Organism Description of Unknown Organism Unknownendonuclease 1 Pro Tyr Val Ile Leu Ile Thr Glu Ile Leu Leu Arg Arg ThrThr 1 5 10 15 2 12 PRT Unknown Organism Description of Unknown OrganismUnknown endonuclease 2 Ala Ile Leu Asp Leu Pro Gly Val Gly Lys Tyr Thr 15 10 3 12 PRT Unknown Organism Description of Unknown Organism Unknownendonuclease 3 Met Val Asp Ala Asn Phe Val Arg Val Ile Asn Arg 1 5 10 475 DNA Artificial Sequence Description of Artificial Sequence SyntheticOligonucleotide 4 aaatggagtt attccaacag ataaagtgtt gaatggaata cttagttatcttggaatgac 60 taaagtagaa ttaga 75 5 74 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Oligonucleotide 5tctaattcta ctttagtaat tccaagataa ttaagtatcc attaaacact ttatttgttg 60gaataactcc attt 74 6 75 DNA Artificial Sequence Description ofArtificial Sequence Synthetic Oligonucleotide 6 tctaatttta ctttagtaattccaagataa ttaagtattc catttaacac tttatatgtt 60 ggaataattc cattt 75 7 34DNA Unknown Organism Description of Unknown Organism NcoI restrictionsite 7 gtggggctgg atttccatgg atgatgctac taat 34 8 30 DNA UnknownOrganism Description of Unknown Organism BamHI site 8 cgacggccagtggatccaag ggggctgatg 30 9 666 DNA Unknown Organism Description ofUnknown Organism ORF 10 9 atg gat gat gct act aat aaa aaa agg aaa gtcttc gtt agc acc ata 48 Met Asp Asp Ala Thr Asn Lys Lys Arg Lys Val PheVal Ser Thr Ile 1 5 10 15 ctt acg ttt tgg aat aca gat agg cgc gac tttcct tgg agg cat acg 96 Leu Thr Phe Trp Asn Thr Asp Arg Arg Asp Phe ProTrp Arg His Thr 20 25 30 agg gac ccc tat gta att tta ata acg gaa atc ctactt cgc agg aca 144 Arg Asp Pro Tyr Val Ile Leu Ile Thr Glu Ile Leu LeuArg Arg Thr 35 40 45 act gcg ggg cat gtt aaa aag ata tat gac aag ttt tttgtt aag tac 192 Thr Ala Gly His Val Lys Lys Ile Tyr Asp Lys Phe Phe ValLys Tyr 50 55 60 aag tgc ttt gag gat ata tta aaa acg cca aaa tca gaa atcgcc aaa 240 Lys Cys Phe Glu Asp Ile Leu Lys Thr Pro Lys Ser Glu Ile AlaLys 65 70 75 80 gac ata aaa gaa atc gga ctc tct aac caa agg gca gaa cagcta aaa 288 Asp Ile Lys Glu Ile Gly Leu Ser Asn Gln Arg Ala Glu Gln LeuLys 85 90 95 gaa ctg gca agg gtc gtc ata aat gat tat ggg ggc aga gtg ccccga 336 Glu Leu Ala Arg Val Val Ile Asn Asp Tyr Gly Gly Arg Val Pro Arg100 105 110 aat agg aag gca att tta gat cta cca gga gtt ggc aaa tac acttgt 384 Asn Arg Lys Ala Ile Leu Asp Leu Pro Gly Val Gly Lys Tyr Thr Cys115 120 125 gct gca gtt atg tgt ttg gca ttt ggc aaa aaa gcc gct atg gtcgat 432 Ala Ala Val Met Cys Leu Ala Phe Gly Lys Lys Ala Ala Met Val Asp130 135 140 gca aat ttt gtg aga gtt att aac agg tac ttt ggg gga agc tatgaa 480 Ala Asn Phe Val Arg Val Ile Asn Arg Tyr Phe Gly Gly Ser Tyr Glu145 150 155 160 aac ctg aac tac aac cac aag gcc ctg tgg gaa ctt gcg gagacc ctt 528 Asn Leu Asn Tyr Asn His Lys Ala Leu Trp Glu Leu Ala Glu ThrLeu 165 170 175 gta cct ggc gga aag tgc agg gac ttt aac ctt ggt tta atggac ttt 576 Val Pro Gly Gly Lys Cys Arg Asp Phe Asn Leu Gly Leu Met AspPhe 180 185 190 tcc gca atc ata tgt gcc cca aga aag cca aag tgt gag aaatgt ggg 624 Ser Ala Ile Ile Cys Ala Pro Arg Lys Pro Lys Cys Glu Lys CysGly 195 200 205 atg agc aaa ctc tgt agc tac tat gag aag tgt agt act tga666 Met Ser Lys Leu Cys Ser Tyr Tyr Glu Lys Cys Ser Thr 210 215 220 10221 PRT Unknown Organism Description of Unknown Organism ORF 10 10 MetAsp Asp Ala Thr Asn Lys Lys Arg Lys Val Phe Val Ser Thr Ile 1 5 10 15Leu Thr Phe Trp Asn Thr Asp Arg Arg Asp Phe Pro Trp Arg His Thr 20 25 30Arg Asp Pro Tyr Val Ile Leu Ile Thr Glu Ile Leu Leu Arg Arg Thr 35 40 45Thr Ala Gly His Val Lys Lys Ile Tyr Asp Lys Phe Phe Val Lys Tyr 50 55 60Lys Cys Phe Glu Asp Ile Leu Lys Thr Pro Lys Ser Glu Ile Ala Lys 65 70 7580 Asp Ile Lys Glu Ile Gly Leu Ser Asn Gln Arg Ala Glu Gln Leu Lys 85 9095 Glu Leu Ala Arg Val Val Ile Asn Asp Tyr Gly Gly Arg Val Pro Arg 100105 110 Asn Arg Lys Ala Ile Leu Asp Leu Pro Gly Val Gly Lys Tyr Thr Cys115 120 125 Ala Ala Val Met Cys Leu Ala Phe Gly Lys Lys Ala Ala Met ValAsp 130 135 140 Ala Asn Phe Val Arg Val Ile Asn Arg Tyr Phe Gly Gly SerTyr Glu 145 150 155 160 Asn Leu Asn Tyr Asn His Lys Ala Leu Trp Glu LeuAla Glu Thr Leu 165 170 175 Val Pro Gly Gly Lys Cys Arg Asp Phe Asn LeuGly Leu Met Asp Phe 180 185 190 Ser Ala Ile Ile Cys Ala Pro Arg Lys ProLys Cys Glu Lys Cys Gly 195 200 205 Met Ser Lys Leu Cys Ser Tyr Tyr GluLys Cys Ser Thr 210 215 220

What is claimed is:
 1. A method of detecting the presence of anddetermining the relative positions of at least two point mutations in atarget polynucleotide, comprising: (a) hybridizing single-strandedoligonucleotide probes to a target polynucleotide to form hybrid,double-stranded polynucleotides such that mismatches occur at the sitesof said point mutations, wherein said probes are complementary to anon-mutated sequence of said target polynucleotide and are labelled atone end but not both ends, and wherein said target polynucleotide is notlabelled; (b) partially digesting the probe strand of said hybridpolynucleotide with a nucleic acid repair enzyme, wherein all possiblerecognition sites within the oligonucleotide probe are not cleaved andprobe fragments of differing lengths are generated; (c) separating saidprobe fragments by size in a medium suitable for visualizing theseparated probe fragments; and then (d) visualizing said separated probefragments in said medium, whereby the presence and relative positions ofsaid point mutations are determined.
 2. The method of claim 1,comprising a further step of measuring the length of said separatedprobe fragments, thereby determining the specific nucleotide position ofeach point mutation.
 3. The method of claim 1, wherein said partialdigestion of step (b) is effected by a nucleic acid repair enzymeselected from the group consisting of muty, T/G mismatch-specificnicking enzyme, human or yeast all-type enzyme, and deoxyinosine3′-endonuclease from E. coli.
 4. The method of claim 1, wherein saidpartial digestion of step (b) comprises one or more additional nucleicacid repair enzymes selected from the group consisting of mutY, T/Gmismatch-specific nicking enzyme, human or yeast all-type enzyme, anddeoxyinosine 3′-endonuclease from E. coli.
 5. The method of claim 1,wherein said partial digestion of step (b) is effected by a nucleic acidrepair enzyme system comprising a glycosylase and a DNA lyase or aglycosylase and a DNA AP endonuclease.
 6. The method of claim 1, whereinsaid partial digestion of step (b) is effected by a thermostable nucleicacid repair enzyme.
 7. The method of claim 6, wherein said thermostablenucleic acid repair enzyme is thymine DNA glycosylase.
 8. The method ofclaim 7, wherein said thymine DNA glycosylase comprises an ORF10 proteinencoded by a DNA sequence comprising the sequence of Seq
 9. 9. Themethod of claim 1, wherein said labeled probe of step (a) isradiolabelled.
 10. The method of claim 1, wherein said separating ofstep (c) is accomplished by gel electrophoresis.
 11. The method of claim1, wherein said target polynucleotide is a cDNA sequence.
 12. A methodof detecting the presence of and determining the relative positions ofat least two point mutations in a target polynucleotide, comprising: (a)hybridizing a single-stranded oligonucleotide probe to a targetpolynucleotide to form a hybrid, double-stranded polynucleotide suchthat mismatches occur at the sites of said point mutations, wherein saidprobe is complementary to a non-mutated sequence of said targetpolynucleotide and is labelled at one end but not both ends, and whereinsaid target polynucleotide is not labelled; (b) partially digesting theprobe strand of said hybrid polynucleotide with a nucleic acid repairenzyme producing oligonucleotide fragments, wherein said oligonucleotideprobe is designed such that said oligonucleotide fragments dissociatefrom said target polynucleotide spontaneously at a predeterminedtemperature; (c) repeating steps (a) and (b) such that probe fragmentsof differing lengths are generated; (d) separating said probe fragmentsby size in a medium suitable for visualizing the separated probefragments; and then (e) visualizing said separated probe fragments insaid medium, whereby the presence and relative positions of said pointmutations are determined.
 13. The method of claim 12, wherein saidhybridization of step (a) is effectuated at a temperature which isapproximately the same as said predetermined temperature ofdissociation.
 14. The method of claim 12, wherein said hybridization ofstep (a) is effectuated at a temperature which is lower than saidpredetermined temperature of dissociation.
 15. The method of claim 12,comprising a further step of measuring the length of said separatedprobe fragments, thereby determining the specific nucleotide position ofeach point mutation.
 16. The method of claim 12, wherein said partialdigestion of step (b) is effected by a nucleic acid repair enzymeselected from the group consisting of muty, T/G mismatch-specificnicking enzyme, human or yeast all-type enzyme, and deoxyinosine3′-endonuclease from E. coli.
 17. The method of claim 12, wherein saidpartial digestion of step (b) comprises one or more additional nucleicacid repair enzymes selected from the group consisting of mutY, T/Gmismatch-specific nicking enzyme, human or yeast all-type enzyme, anddeoxyinosine 3′-endonuclease from E. coli.
 18. The method of claim 12,wherein said partial digestion of step (b) is effected by a nucleic acidrepair enzyme system comprising a glycosylase and a DNA lyase or aglycosylase and a DNA AP endonuclease.
 19. The method of claim 12,wherein said partial digestion of step (b) is effected by a thermostablenucleic acid repair enzyme.
 20. The method of claim 19, wherein saidthermostable nucleic acid repair enzyme is thymine DNA glycosylase. 21.The method of claim 20, wherein said thymine DNA glycosylase comprisesan ORF10 protein encoded by a DNA sequence comprising the sequence ofSeq.
 9. 22. The method of claim 12, wherein said labeled probe of step(a) is radiolabelled.
 23. The method of claim 12, wherein saidseparating of step (d) is accomplished by gel electrophoresis.
 24. Themethod of claim 12, wherein said target polynucleotide is a cDNAsequence.
 25. The method of claim 12, wherein a heat destabilizationmolecule is added to said hybridizing of step (a), such that thetemperature of hybridization is decreased.